Alternative splicing provides a mechanism for the production of related yet distinct proteins from a single gene transcript, thus generating diversity. Gene therapy based on alternative splicing of gene sequences should mimic the full range of physiological effects more closely than the use of a single cDNA, likely resulting in increased efficacy and reduced adverse effects. In the case of angiogenic gene therapy using vascular endothelial growth factor (VEGF), all human studies have used single cDNAs. However, physiological angiogenesis occurs by the concerted action of at least three major isoforms, 121, 165 and 189; these isoforms have subtly different properties and are produced in different ratios in various tissues. The focus of this proposal is to incorporate the concept of alternative splicing into the paradigm of gene therapy, thus more accurately reflecting the true phenotypic potential of the transferred gene. The underlying hypothesis of this proposal, is that VEGF genes that direct the production of mixtures of isoforms will enhance the effectiveness of VEGF-mediated angiogenesis while reducing the adverse consequences resulting from the use of a single cDNA that expresses a single isoform. Although the proposed studies focus only on the VEGF gene, and utilize only adenovirus (Ad) gene transfer vectors, the basic principles that will be generated should be applicable to any gene and any gene transfer vector. With this background, the specific aims of the proposal include: Specific Aim 1. Engineer genomic/cDNA hybrid genes that express different ratios of the major VEGF isoforms and demonstrate in vitro and in vivo that gene transfer with these constructs results in different patterns of cell- and tissue-specific alternative splicing. Specific Aim 2. Assess splicing patterns of the hybrid genomic/cDNA VEGF genes when targeted to express in different cell types of the same organ. Specific Aim 3. Evaluate the efficacy vs adverse phenotypes of gene transfer resulting in different mixtures of VEGF isoforms following in vivo transfer of VEGF genomic/cDNA hybrids designed to generate different patterns of alternative splicing.